Pyrazolo[1,5-a]pyrimidine based Trk inhibitors: Design, synthesis, biological activity evaluation

Article abstract of DOI:10.1016/j.bmcl.2020.127712

Tropomyosin receptor kinases (Trks), a transmembrane receptor tyrosine kinases, have attracted more and more attention as a drug target. Here we reported the structure-based synthesis and biological evaluation of novel pyrazolo[1,5-a]pyrimidine derivatives as Trk inhibitors, which exhibited potent Trk inhibitory activities. Particularly, compounds 8a, 8f, 9a, 9b and 9f (IC₅₀ < 5 nM) showed significant inhibitory potency against Trk.

Full text of DOI:10.1016/j.bmcl.2020.127712

Journal Pre-proofs
Pyrazolo[1,5-a]pyrimidine Based Trk Inhibitors: Design, Synthesis, Biologi‐
cal Activity Evaluation
Yongjie Zhang, Yan Liu, Ying Zhou, Qing Zhang, Tianfu Han, Chunlei Tang,
Weizheng Fan
PII:
S0960-894X(20)30823-4
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https://doi.org/10.1016/j.bmcl.2020.127712
BMCL 127712
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17 November 2020
20 November 2020
Please cite this article as: Zhang, Y., Liu, Y., Zhou, Y., Zhang, Q., Han, T., Tang, C., Fan, W., Pyrazolo[1,5-
a]pyrimidine Based Trk Inhibitors: Design, Synthesis, Biological Activity Evaluation, Bioorganic & Medicinal
Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127712
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Pyrazolo[1,5-a]pyrimidine Based Trk Inhibitors: Design, Synthesis,
Biological Activity Evaluation.
Yongjie Zhang†, Yan Liu†, Ying Zhou, Qing Zhang, Tianfu Han, Chunlei Tang*, Weizheng Fan*.
School of Pharmaceutical Science, Jiangnan University, Wuxi, China.
†
These authors contributed equally to this work
*Corresponding
author.
E-mail
address:
tangcl@jiangnan.edu.cn
(C.
Tang);
wzfan@jiangnan.edu.cn (W. Fan)
ABSTRACT:
Tropomyosin receptor kinases (Trks), a transmembrane receptor tyrosine kinases, have attracted
more and more attention as a drug target. Here we reported the structure-based synthesis and
biological evaluation of novel pyrazolo[1,5-a]pyrimidine derivatives as Trk inhibitors, which
exhibited potent Trk inhibitory activities. Particularly, compounds 8a, 8f, 9a, 9b and 9f (IC50<5
nM) showed significant inhibitory potency against Trk.
Keywords: Tropomyosin receptor kinases; Anticancer; Design and synthesis; Molecular docking
The three members of the tropomyosin receptor kinase (Trk) family, namely TrkA (encode by
NTRK1), TrkB (NTRK2) and TrkC (NTRK3), belong to receptor tyrosine kinases (RTKs), which
1,
2
are transmembrane proteins.
Mainly expressed in neuronal tissues and activated by
neurotrophins, these kinases take an essential role in regulating cell survival, proliferation, the fate
of neural precursors, axon and dendrite growth and patterning.1-4 NTRK rearrangement and fusion
have been found in varying proportions in a broad array of tumors types.5, 6 NTRK gene fusion,
point mutation, and in-frame deletions produce a ligand-independent constitutively activated
protein with carcinogenic potential.7-9 Due to the structural or functional loss of the extracellular
domain, these genetic abnormalities are difficult to be regulated by means such as antibody
therapy10, while the small-molecule inhibitors have become a promising approach.11, 12
There are several Trk inhibitors in clinical trials, two of which, Larotrectinib and Entrectinib,
have got gratifying progress.13 In basket trials, Trk inhibitors (such as Larotrectinib and
Entrectinib) have remarkable and durable antitumor activity in patients with diverse cancers
harbouring oncogenic fusions involving the neurotrophin receptor tyrosine kinase genes NTRK1,
NTRK2 or NTRK3.14, 15 These successes have led to more interests in small molecule inhibitors
against Trk.
The Trk inhibitors of type I, such as Larotrectinib and Entrectinib, have rich molecular
diversity, but most of them interact with the Met (Met592 in TrkA, Met636 in TrkB, Met620 in
TrkC) in the hinge region at the ATP site.16 Based on the molecular docking model (Fig. 1) and the
structure similarities between Larotrectinib and GNF-8625,16, 17 it could be assumed that the N1 of
pyrazolo[1,5-a]pyrimidine of Larotrectinib also had hydrogen bond interaction with Met in the

hinge region of Trk. In addition, it could be seen from the docking results that this heterocyclic
core was critical for placing the difluorophenyl and 3-pyrrolidinol groups in an appropriate spatial
position. Therefore, such a scaffold might not be replaceable. However, given the rigidity of the
pyrrole ring, which links the heterocyclic scaffold and difluorophenyl, it’s decyclization could be
more conducive to placing the difluorophenyl in the hydrophobic pocket. Besides, the
3-pyrrolidinol groups extending towards the leading edge of the solvent might be underutilized in
this polar region. Perhaps the introduction of some other group (R1 in Fig. 2) would result in
interesting changes in activity that could help to elucidate the SAR of compounds with such
scaffold structures.
Based on the above analysis, we designed and synthesized a series of new chemical entities
(Fig.2). The synthetic approach to these compounds (Scheme 1.) started from commercially
available
2,
5-difluorophenone
(compound
1).
Nucleophilic
addition
with
(R)-2-methylpropane-2-sulfinamide and then reduction by sodium borohydride gave rise to
sulfonamide derivatives (compound 2). The R- tert-butyl sulfonyl group was removed in a dioxane
solution of hydrogen chloride to afford (R)-1-(2, 5-difluorophenyl) ethan-1-amine (compound 3).
Nucleophilic addition with formaldehyde solution and then reduction by sodium borohydride gave
the (R)-1-(2, 5-difluorophenyl)-N-methylethan-1-amine (compound 4). The nucleophilic
substitution reaction of commercial material (compound 5) with 3 resulted in 6a, or with 4
resulted in 6b. Then, nitrification was performed under the action of trifluoroacetic acid and nitric
acid followed by reduction with zinc to obtain intermediate 7a and 7b. Reaction with N, N'
-carbonyl diimidazole and amines of different structures produced two series of urea derivant 8a-h
and 9a-h. Material 10 and 11 undergo a transesterification to get compound 12, and then 13a and
13b were obtained by by amine ester exchange reaction of 12 with 7a and 7b, respectively.
Thiocarbamides 14a or 14b was afford by reaction of 7a or 7b with the thiophosgene followed by
nucleophilic addition reaction with (S) -3-hydroxypyrrolidine hydrochloride.
Fig.1. The docking model of Larotrectinib binding TrkA; Hydrogen bonds with Met592 are shown
as bright green lines. The kinase is depicted in offwhite ribbons. Compound atoms are colored as
follows: carbon, yellow; nitrogen, blue; oxygen, red; fluorine, cyan; sulfur, orange.

Fig.2. Design and modification of a new chemical entity based on pyrazolo[1,5-a]pyrimidine
structure
Scheme 1. Synthesis of compounds 8a-h, 9a-h, 13a-13b, 14a-14b. Reagents and conditions: (i)
(R)-2-methylpropane-2-sulfinamide, Ti(OEt)4, THF, 70 oC, 2 h,94%; NaBH4, THF, H2O, -50 oC to
R.T., 3.5 h,84%; (ii) HCl in 1,4-dioxane, R.T., overnight,78%; (iii) HCHO, NaBH4, MeOH, R.T.,
1 h,87%; (iv) DIPEA, n-BuOH, 160 oC, 4 h,60%-65%; (v) HNO3, TFA, R.T., 0.5 h,55%-88%; Zn
dust, NH4Cl, MeOH, DCM, R.T., 0.5 h,90%; (vi) CDI, TEA , DCM, R.T., 2.5 h,20%-60%; (vii)
TEA, EtOH, R.T., 4 h,80%; (viii) Zn(OTf)2, EtOH, 80 oC, overnight, 36%-50%; (ix) CCl2S, TEA,
DCM, 0oC to R.T., 1 h; TEA, THF, R.T., 2 h,50%-55%.
All synthesized compounds were evaluated for inhibitory activity against TrkA, TrkB and
TrkC, taking Larotrectinib as a positive control. Table 1 summarized the structure-activity
relationship (SAR) of derivatives modified at the 5-position and 3-position of
pyrazolo[1,5-a]pyrimidine. Firstly, we introduced the (R)-1-(2, 5-difluorophenyl) ethan-1-amine
and (R)-1-(2, 5-difluorophenyl)-N-methylethan-1-amine at the 5-position of the scaffold.

Decyclization of the pyrrole ring linker showed no noteworthy influence on activity against Trk,
through comparing compounds 8a (IC50=1.9 nM, 3.1 nM, 2.3 nM against TrkA, TrkB, TrkC,
respectively) or 9a (IC50=1.8 nM, 4.1 nM, 2.3 nM) with positive control (IC50=1.2 nM, 2.1 nM,
2.1 nM).
Then we focused on introducing different moieties containing hydroxyl at the 3-position. All
the compounds (8b-e, 9b-e), with the exception of 9b, displayed reduced inhibitory potency for
Trk, when compared with compound 8a or 9a. It seemed that piperidin-3-ylmethanol,
azetidin-3-ol, 2-aminobutan-1-ol were not as good as 3-pyrrolidinol groups at generating
beneficial interactions with the solvent exposed region. However, compound 9b (IC50=2.5 nM, 3.1
nM, 2.6 nM against TrkA, TrkB, TrkC, respectively) exhibited more than 20 fold stronger
inhibitory activity than 8b (IC50=56 nM, 89 nM, 53 nM). This difference could be reasonably
explained by structural modeling. In the docking models of 8b (Fig.3A) and 9b (Fig.3B),
compared to the Larotrectinib (Fig. 1), the orientation of the heterocyclic core was flipped, placing
the hydroxyphenyl ring in the hydrophobic pocket originally occupied by Phe669 instead of
difluorophenyl. Similar interesting situation in X-ray cocrystal structure have been reported in
previous studies.16, 17And methylation of the benzylic amine led to powerful π-π interaction
between difluorophenyl of 9b and Phe521, which might cause the significant difference of this
two compounds.
Interestingly, compounds 8f (IC50=3.2 nM, 5.5 nM, 3.3 nM against TrkA, TrkB, TrkC,
respectively) and 9f (IC50=1.4 nM, 2.4 nM, 1.9 nM) showed comparable activity to positive
control when the 3-position ureas were substituted by hydrophobic diisopropyl. Then, to better
understand the mechanism of inhibition, we hypothesized that, similar to the computer modeling
of 9b, in the binding structure of 8f or 9f to kinase protein, the reduction of free energy generated
by placing diisopropyl in hydrophobic pocket was the main cause of strong kinase inhibitory
effect. Subsequent computational efforts also corroborated this hypothesis (Supplemental Fig.1).
When we designed to replace the hydroxyl moiety with aliphatic amines, both cyclic (8g, 9g)
and chain (8h, 9h) fatty amines showed reduction in inhibitory efficacy. However, the 10-fold
difference in inhibitory activity between 9h and 8h caught our attention. By modeling study,
similar to 9b and 8b, the orientation of the 8h’s scaffold was reversed too, while 9h’s scaffold
orientation was consistent with 8a and 9a (Supplemental Fig.2). Finally, we turned our attention to
the modification of urea groups, and got compounds 13a-b and 14a-b. A larger group replacing
the carbonyl of the urea resulted in worse performance for 13a-b. And when sulfur atoms were
substituted for oxygen atoms, 14a-b showed bad inhibitory activity against Trk.
In general, as indicated by the results, when R2 is (R)-1-(2, 5-difluorophenyl)
-N-methylethane-1-amine, compared with the corresponding N-unmethylated compounds, the
inhibitory activity against Trk is enhanced to varying degrees. Based on the previous analysis, it
can be seen that the linker between difluorophenyl and heterocyclic nucleus is in the hydrophobic
region, regardless of how the scaffold of the compound place the bilateral moiety, which
contributes to increased potency. Finally, throughout the series, all compounds showed no
significant selectivity against TrkA, TrkB, and TrkC.

Fig. 3. (A) The docking model of 8b binding TrkA. (B) The docking model of 9b binding TrkA.
The kinase is depicted in offwhite ribbons. The π-π interaction are illustrated with red lines, and
the hydrogen bonds with Met592 are shown as green lines. Compound atoms are colored as
follows: carbon, yellow; nitrogen, blue; oxygen, red; fluorine, cyan; sulfur, orange.
Table 1
In Vitro Trk inhibitory activities of the designed compounds.
ID
8a
R1
R2
IC50a(nM)TrkA IC50a(nM)TrkB IC50a(nM)TrkC
1.9
56
15
71
89
3.2
3.1
2.3
53
21
82
78
3.3
8b
8c
8d
8e
8f
89
32
102
153
5.5

8g
8h
112
126
360
230
221
490
151
145
412
13a
14a
9a
54
1.8
2.5
18
87
4.1
3.1
31
51
2.3
2.6
25
9b
9c
9d
101
146
92
9e
9f
42
57
36
1.4
2.4
1.9

9g
9h
21
8.0
62
38
14
87
26
16
39
13b
14b
19
28
19
Larotrectinib
1.2
2.1
2.1
Bold values represent compounds that are found better inhibitory activity.
a
Values are means of at least two or more experiments. Standard deviations were<10% of the
mean.
Conclusions
In summary, we have designed and synthesized a series of novel pyrazolo[1,5-a]pyrimidine
derivatives as Trk inhibitors. The biochemical activity of all compounds against Trks were
reported in Tables 1. The brief SAR study demonstrated that the compounds with
(R)-1-(2,5-difluorophenyl) -N-methylethane-1-amine substituent at the 5-position (9a-h,13a,14a)
had better inhibitory potency in comparison with the corresponding N-unmethylated compounds
(8a-h,13b,14b). Derivatives 8a, 8f, 9a, 9b and 9f were proved to be the best compounds of the
series, showing remarkable activity on Trks (IC50< 5 nM), which was comparable to Larotrectinib.
Especially, initial mechanism study, which was elucidated by docking model (8f, 9b and 9f),
indicated that putting polar groups oriented toward the solvent front may not be necessary. So we
had a hypothesis that inhibitors with such binding mode would be still valid to Trk with
solvent-front mutations (SFM), which often occurred after treatment with Larotrectinib and
Entrectinib.18-21 Further studies to explore the mechanism of these compounds are needed and now
in progress. The evolution of these series of compounds will be reported in due course.
Acknowledgements
This work was supported by the National First-class Discipline Program of Food Science and
Technology (JUFSTR20180101) and the National Natural Science Foundation of China (Grant
Nos. 21305051).

1. Huang, E. J.;Reichardt, L. F., Trkreceptors:Rolesinneuronalsignaltransduction.
Annu. Rev. Biochem. 2003, 72, 609-642.
2. Carter, A. R.; Chen, C. F.; Schwartz, P. M.; Segal, R. A., Brain-derived
neurotrophic factor modulates cerebellar plasticity and synaptic ultrastructure.
Journal of Neuroscience 2002, 22 (4), 1316-1327.
3. Bartkowska, K.; Paquin, A.; Gauthier, A. S.; Kaplan, D. R.; Miller, F. D., Trk
signaling regulates neural precursor cell proliferation and differentiation during
cortical development. Development 2007, 134 (24), 4369-4380.
4. Joshi, S. K.; Davare, M. A.; Druker, B. J.; Tognon, C. E., Revisiting NTRKs as
an emerging oncogene in hematological malignancies. Leukemia 2019, 33 (11), 2563-2574.
5. Amatu, A.; Sartore-Bianchi, A.; Siena, S., NTRK gene fusions as novel targets of
cancer therapy across multiple tumour types. Esmo Open 2016, 1 (2).
6. Vaishnavi, A.; Le, A. T.; Doebele, R. C., TRKing Down an Old Oncogene in a New
Era of Targeted Therapy. Cancer Discovery 2015, 5 (1), 25-34.
7. Coulier, F.; Kumar, R.; Ernst, M.; Klein, R.; Martin-Zanca, D.; Barbacid, M.,
Human trk oncogenes activated by point mutation, in-frame deletion, and duplication of
the tyrosine kinase domain. Molecular and cellular biology 1990, 10 (8), 4202-10.
8. Farago, A. F.; Le, L. P.; Zheng, Z.; Muzikansky, A.; Drilon, A.; Patel, M.;
Bauer, T. M.; Liu, S. V.; Ou, S.-H. I.; Jackman, D.; Costa, D. B.; Multani, P. S.;
Li,G.G.; Hornby,Z.; Chow-Maneval,E.; Luo,D.; Lim,J.E.; Iafrate,A.J.;Shaw,
A. T., Durable Clinical Response to Entrectinib in NTRK1-Rearranged Non-Small Cell Lung
Cancer. Journal of Thoracic Oncology 2015, 10 (12), 1670-1674.
9. Tacconelli, A.; Farina, A. R.; Cappabianca, L.; DeSantis, G.; Tessitore, A.;
Vetuschi, A.; Sferra, R.; Rucci, N.; Argenti, B.; Screpanti, I.; Gulino, A.;
Mackay, A. R., TrkA alternative splicing: A regulated tumor-promoting switch in human
neuroblastoma. Cancer Cell 2004, 6 (4), 347-360.
10. Segal, R. A., Selectivity inneurotrophin signaling: Theme and variations. Annual
Review of Neuroscience 2003, 26, 299-330.
11. Mitra, G.; Martin-Zanca, D.; Barbacid, M., Identification and biochemical
characterization of p70TRK, product of the human TRK oncogene. Proceedings of the
National Academy of Sciences of the United States of America 1987, 84 (19), 6707-11.
12.Greco, A.; Fusetti, L.; Miranda, C.; Villa, R.; Zanotti, S.; Pagliardini, S.;
Pierotti, M. A., Role of the TFG N-terminus and coiled-coil domain in the transforming
activity of the thyroid TRK-T3 oncogene. Oncogene 1998, 16 (6), 809-816.
13. Roviello, G.; D'Angelo,A.; Sciortino, M.; Mini, E.; Nobili,S.; DeLogu, F.;
Massi, D., TRK fusion positive cancers: From first clinical data of a TRK inhibitor to
future directions. Critical Reviews in Oncology Hematology 2020, 152.
14.Drilon, A.; Laetsch, T. W.; Kummar, S.; DuBois, S. G.; Lassen, U. N.; Demetri,
G. D.; Nathenson, M.; Doebele, R. C.; Farago, A. F.; Pappo, A. S.; Turpin, B.;
Dowlati, A.; Brose, M. S.; Mascarenhas, L.; Federman, N.; Berlin, J.; El-Deiry,
W. S.; Baik, C.; Deeken, J.; Boni, V.; Nagasubramanian, R.; Taylor, M.;
Rudzinski, E. R.; Meric-Bernstam, F.; Sohal, D. P. S.; Ma, P. C.; Raez, L. E.;
Hechtman, J. F.; Benayed, R.; Ladanyi, M.; Tuch, B. B.; Ebata, K.; Cruickshank,
S.; Ku, N. C.; Cox, M. C.; Hawkins, D. S.; Hong, D. S.; Hyman, D. M., Efficacy of

Larotrectinib in TRK Fusion-Positive Cancers in Adults and Children. N. Engl. J. Med.
2018, 378 (8), 731-739.
15. Drilon, A.; Siena, S.; Ou, S.-H. I.; Patel, M.; Ahn, M. J.; Lee, J.; Bauer,
T. M.; Farago, A. F.; Wheler, J. J.; Liu, S. V.; Doebele, R.; Giannetta, L.;
Cerea, G.; Marrapese, G.; Schirru, M.; Amatu, A.; Bencardino, K.; Palmeri, L.;
Sartore-Bianchi, A.; Vanzulli, A.; Cresta, S.; Damian, S.; Duca, M.; Ardini, E.;
Li, G.; Christiansen, J.; Kowalski, K.; Johnson, A. D.; Patel, R.; Luo, D.;
Chow-Maneval, E.; Hornby, Z.; Multani, P. S.; Shaw, A. T.; De Braud, F. G., Safety
andAntitumorActivityoftheMultitargetedPan-TRK, ROS1, andALKInhibitorEntrectinib:
Combined Results from Two Phase I Trials (ALKA-372-001 and STARTRK-1). Cancer Discovery
2017, 7 (4), 400-409.
16. Yan, W.; Lakkaniga, N. R.; Carlomagno, F.; Santoro, M.; McDonald, N. Q.; Lv,
F.; Gunaganti, N.; Frett, B.; Li, H.-y., Insights into Current Tropomyosin Receptor
Kinase (TRK) Inhibitors: Development and Clinical Application. Journal of Medicinal
Chemistry 2019, 62 (4), 1731-1760.
17. Choi, H.-S.; Rucker, P. V.; Wang, Z.; Fan, Y.; Albaugh, P.; Chopiuk, G.;
Gessier, F.; Sun, F.; Adrian, F.; Liu, G.; Hood, T.; Li, N.; Jia, Y.; Che, J.;
McCormack, S.; Li, A.; Li, J.; Steffy, A.; Culazzo, A.; Tompkins, C.; Van, P.;
Kreusch, A.; Lu, M.; Hu, B.; Chaudhary, A.; Prashad, M.; Tuntland, T.; Liu, B.;
Harris, J.; Seidel, H. M.; Loren, J.; Molteni, V., (R)-2-Phenylpyrrolidine
Substituted Imidazopyridazines: A New Class of Potent and SelectivePan-IRK Inhibitors.
Acs Med Chem Lett 2015, 6 (5), 562-567.
18. Parikh, A. R.; Corcoran, R. B., Fast-TRKing Drug Development for Rare Molecular
Targets. Cancer Discovery 2017, 7 (9), 934-936.
19. Drilon, A.; Nagasubramanian, R.; Blake, J. F.; Ku, N.; Tuch, B. B.; Ebata,
K.; Smith,S.; Lauriault,V.; Kolakowski,G.R.; Brandhuber,B.J.; Larsen,P.D.;
Bouhana, K. S.; Winski, S. L.; Hamor, R.; Wu, W.-I.; Parker, A.; Morales, T. H.;
Sullivan, F. X.; DeWolf, W. E.; Wollenberg, L. A.; Gordon, P. R.; Douglas-Lindsay,
D. N.; Scaltriti, M.; Benayed, R.; Raj, S.; Hanusch, B.; Schram, A. M.; Jonsson,
P.; Berger, M. F.; Hechtman, J. F.; Taylor, B. S.; Andrews, S.; Rothenberg, M.;
Hyman, D. M., A Next-Generation TRK Kinase Inhibitor Overcomes Acquired Resistance to
Prior TRK Kinase Inhibition in Patients with TRK Fusion-Positive Solid Tumors. Cancer
Discovery 2017, 7 (9), 963-972.
20. Russo, M.; Misale, S.; Wei, G.; Siravegna, G.; Crisafulli, G.; Lazzari, L.;
Corti, G.; Rospo, G.; Novara, L.; Mussolin, B.; Bartolini, A.; Cam, N.; Patel,
R.; Yan, S.; Shoemaker, R.; Wild, R.; Di Nicolantonio, F.; Sartore-Bianchi, A.;
Li, G.; Siena, S.; Bardelli, A., Acquired Resistance to the TRK Inhibitor Entrectinib
in Colorectal Cancer. Cancer Discovery 2016, 6 (1), 36-44.
21. Fuse, M. J.; Okada, K.; Oh-hara, T.; Ogura, H.; Fujita, N.; Katayama, R.,
Mechanisms of Resistance to NTRK Inhibitors and Therapeutic Strategies in
NTRK1-Rearranged Cancers. Molecular Cancer Therapeutics 2017, 16 (10), 2130-2143.

Highlights
• Novel pyrazolo[1,5-a]pyrimidine derivatives were designed and synthesized.
• TrkA, TrkB and TrkC inhibitory activity were evaluated.
• Compounds 8a, 8f, 9a, 9b and 9f (IC50<5 nM) showed significant inhibitory potency against Trk.